Project Summary/Abstract The long-term goals of the proposed research program are to provide insight into the complex dialogue that has evolved between humans and their normal beneficial microbiota. Recent research has indicated a strong correlation between the successful maintenance of this dialog and life-long health. However, the inaccessibility of colonized tissues in mammals and the high diversity of their microbiomes render an in-depth study of persistent colonization of the human gut track extremely challenging. When faced with such complex phenomena, biologists often turn to simpler model systems to provide insights into evolutionarily conserved features and reveal basic principles. To decipher the cellular and molecular mechanisms underlying the chronic association of bacteria with apical surfaces of mucosal epithelia, the proposed program exploits the specific, binary symbiosis between the bacterium Vibrio fischeri and its squid host, Euprymna scolopes. This discrete, experimentally tractable, association has been studied for almost three decades as a model for the long-term colonization of mucosa by Gram-negative bacteria. As in humans, the squid-vibrio association begins anew each generation, and fosters the continuing health of both partners. The symbiosis can be initiated with V. fischeri strains with defined mutations, and the host can be bred and maintained for its entire life. The association can be directly imaged using confocal microscopy, which offers the rare opportunity to define, with high temporal and spatial resolution, the reciprocal molecular and genetic dialogue in a life-long beneficial association. This project combines the expertise of the two co-PIs, each with experience in the biology of one of the symbiotic partners, with additional analytical expertise of three collaborators. Together, they introduce new approaches and technology to the study of host-microbe interactions, including: reciprocal epigenomic analyses of the effect of symbiosis on both partners; hybridization-chain-reaction, fluorescent in situ hybridization (HCR-FISH), which enables visualization of rare transcripts in both host and symbiont cells in colonized tissues; NanoString Technology, a new method for simultaneous analysis of dozens to hundreds of targeted transcripts; and, high-efficiency RNAseq, which produces robust transcriptional libraries from as little as 10 ng total RNA (~100,000 bacteria). Specific aims to be addressed are: (i) determining how a daily metabolic rhythm is coordinated between a host and its microbiome; (ii) discovering how the symbionts maintain a rhythmic cycle of growth and persistence; and, (iii) defining the role of epigenetic modifications in the development and maintenance of a beneficial symbiosis. An understanding of the human microbiome is in its infancy, and this frontier field is currently at the stage of building paradigms. Within this context, as the squid-vibrio system has in the past, the results of the current study will shed light upon fundamental principles that govern persistent colonization by both beneficial and pathogenic microbes.